Causal or not: applying the bradford hill aspects of evidence to the association between zika virus and microcephaly

Causal or not applying the Bradford Hill aspects of evidence to the association between Zika virus and microcephaly Opinion Causal or not applying the Bradford Hill aspects of evidence to the associat[.]

Causal or not: applying the Bradford Hill

aspects of evidence to the association

between Zika virus and microcephaly

Christina Frank, Mirko Faber & Klaus Stark

After the emergence of unusual clusters

of microcephaly among babies born

in the fall of 2015 in Brazil—and,

retrospectively, in French Polynesia—

WHO declared a Public Health Emergency of

International Concern in January 2016 (http://

www.who.int/mediacentre/news/statements/

2016/1st-emergency-committee-zika/en/)

Based on the spatial and temporal

correla-tion of these clusters with outbreaks of Zika

virus infections a few months before, a causal

link is suspected Whether the observed link

between infection and microcephaly is indeed

causal needs careful assessment—not the

least because we might fall into the trap of

ecological fallacy, inferring a causal

tion on the individual level from an

associa-tion on the aggregate or populaassocia-tion level

Here, we organize the currently available

data into Austin Bradford Hill’s aspects of

evidence for the consideration of causality

(Hill, 1965) Although it is no longer

consid-ered a litmus test in itself, Hill’s recipe for

careful and multifaceted contemplation of

the available evidence is still a rigorous

method for separating what is known from

what is not known For each aspect, we first

consider individual-level evidence, but

supplement it with likely more readily

observable effects on the population level

Strength of the association

Studies to characterize the strength of this

association in terms of odds ratios from

case–control studies or (preferable) relative

risks from cohort studies are already

underway These should be able to answer

how much more likely a mother infected

with Zika virus any time during pregnancy,

or in a specific time window, will be giving birth to a child with microcephaly compared

to mothers who were not infected On the ecological level, a strong increase in micro-cephaly cases is not by itself evidence for a strong causal association because there might

be confounding or biases Nevertheless, if there were a strong causal association, we would expect to see a large increase in cases

of microcephaly in a population with many Zika virus infections during pregnancy

Consistency

If the association is causal, maternal Zika virus infection would consistently predict a risk of microcephaly in the fetus or newborn But such data are not available yet Isolated cases of congenital micro-cephaly and Zika virus infection outside the outbreak areas, such as the travel-associated cases on Hawaii and in Slovenia, provide some limited evidence toward a consistent association regardless of area of permanent residence (Mlakar et al, 2016)

On the ecological level, the incidence of microcephaly apparently increased in temporal and spatial association with virus outbreaks in Brazil and French Polynesia—

two out of two countries with larger popula-tions, for which the outcomes of early preg-nancies potentially affected during the virus outbreak are already known However, this

is not yet the case in many other countries

in the Americas that experienced Zika virus outbreaks in recent months Thus, the current lack of microcephaly clusters there does not argue against causality

On first glance, it is inconsistent and even

a bit irritating that outbreaks of micro-cephaly have not been observed in other parts of the world that likely are endemic for Zika virus, namely tropical areas in Africa and Asia Potential reasons include high infant mortality, and patchy perinatal care and surveillance systems In addition, endemic Zika virus infection could generate high levels of immunity within the popula-tion: If most people were infected during childhood, women of child-bearing age would be largely immune and congenital infections rare To ascertain the status of infection and immunity in endemic regions requires serosurveys using diagnostic tools that can reliably differentiate between anti-bodies to the various flaviviruses, especially dengue virus If the populations in tropical Africa and Asia are indeed largely immune, the association between Zika virus and microcephaly may only have come to light

in Brazil, because the virus, uncommonly, met an immunologically naı¨ve population of substantial size in a country with surveillance for congenital birth defects

Specificity

Specificity is always a problematic aspect for diseases that may have more than one cause A causal relationship between Zika virus infection during pregnancy and micro-cephaly of the child cannot be specific, because other causes for microcephaly abound, including infections with cytomega-lovirus, rubella virus, or Toxoplasma gondii However, it may be possible to determine a certain type of impact on the developing

Department for Infectious Disease Epidemiology, Robert Koch Institute, Berlin, Germany E-mail: frankc@rki.de

DOI 10.15252/emmm.201506058 | Published online 14 March 2016

ª 2016 The Authors Published under the terms of the CC BY 4.0 license EMBO Molecular Medicine Vol 8 | No 4 | 2016 305

brain that is specific to Zika virus This

would be important evidence for a causal

association

Temporality

Many individual cases show the right order

of Zika virus infection of the mother and

microcephaly in the newborn Detailed data

on this temporal association will also help to

identify the specific time window during

pregnancy when the fetus’ developing brain

would be vulnerable to the virus On the

population level, we can see the same

temporal pattern of microcephaly clusters

following Zika virus outbreaks in French

Polynesia and in Brazil If temporality

holds, we would expect an increase in

congenital microcephaly in summer 2016 in

the other countries that have been

appar-ently affected by the virus since late 2015/

early 2016

Biological gradient

Infection is a binary condition, and “dose–

response”-type evidence is therefore very

rare in this context However, there may be

a graded association between the level of

viremia in the mother or the severity of her

symptoms and the probability that the child

is born with microcephaly Specific

pathological data associated with Zika virus

infection at certain phases during pregnancy—

akin to “the earlier in pregnancy the mother

is infected, the worse the grade of

micro-cephaly”—could be interpreted as somewhat

analogous evidence On the population

level, if the association is causal, we should

see a stronger increase in the incidence of

microcephaly where more pregnant women

were potentially infected with Zika virus

Plausibility

A number of infectious agents are known to

interfere with morphogenetic processes in

the embryo or fetus during the blastogenesis

period, resulting in congenital

malforma-tions owing to cytotoxic effects or inhibition

of mitosis In the case of Zika virus, various

reports found viral RNA and antigens in

amniotic fluid of infected mothers and the

brains of microcephaly-affected fetuses (e.g.,

Mlakar et al, 2016) and newborns who died

after birth (e.g., Martines et al, 2016) These

reports demonstrate congenital Zika virus

infection, including penetration of the

placenta and the fetal blood–brain barrier

Based on what is already known about the virus, it appears to have neuropathological properties (as discussed by Tetro, 2016), which would fit findings of fetal micro-cephaly such as in the comprehensively investigated case in Slovenia (Mlakar et al, 2016) Since the specific malformations appeared typical for an infectious cause, and since other infectious causes of micro-cephaly were actively ruled out, this individ-ual case clearly points toward a causal role

of Zika virus

Coherence

There is some, albeit seasoned, information that Zika is a neurotropic virus in experi-mental animals (Dick et al, 1952; Bell et al, 1971) Further studies in animal models are just beginning If Zika virus infection in the gravid animal model would show associa-tions with microcephaly and comparable changes of the central nervous system (CNS)

in the offspring, or if matching neuropatho-logical effects of the virus could be observed

in cell culture, it would further support causality Given that there are other viral infections already known to be causally associated with microcephaly and other changes of the CNS, adding Zika virus to the list would not violate any established scien-tific concepts

Experiment

Regarding the association between Zika virus and microcephaly, this aspect only pertains to data from animal models or

“natural” experiments on the population level In terms of the latter, it will be impor-tant and interesting to study differences in the association between virus infection and microcephaly between different population groups in Brazil—the poor and the affluent,

or those who sought treatment and those who did not, or those with additional expo-sures or not The role of previous exposure

to other viruses and resulting effects on the immune system should also be studied This will help to identify other, potentially rele-vant factors influencing the association between maternal Zika virus infection and congenital microcephaly In addition, more cases similar to the Slovenian case—congen-ital microcephaly with evidence of prior maternal infection during brief periods of travel to outbreak areas—would argue

against a role of potential (co-)factors speci-fic to being pregnant in Brazil and other affected areas

Analogy

There is some evidence that a number of other flaviviruses can cause congenital brain malformations in humans and animals Among the animal pathogenic flaviviruses, Wesselsbron or Japanese encephalitis viruses are known to cause teratogenic effects (for details, see Tsai, 2006) During the large West Nile virus (WNV) epidemics in North America, one case of congenital infection with resulting CNS damage was reported (Alpert et al, 2003) But more extensive stud-ies of infants whose mothers became infected during pregnancy did not demonstrate a large risk of congenital infection and

“outbreaks” of brain malformations trailing the WNV outbreaks were not identified Causal links specific to microcephaly are established for viruses from other families, such as Cytomegalovirus or rubella virus The analogy to rubella is particularly intrigu-ing: Rubella virus is a Togavirus and maternal infections during pregnancy can cause congenital rubella syndrome (CRS) in the unborn child Among the manifestations

of CRS is microcephaly with cerebral calcifi-cations (Katz et al, 1968), as has been described in many apparently Zika virus-associated microcephaly cases (Mlakar et al, 2016) Before vaccines became available, rubella epidemics and even pandemics resulted in clusters of CRS whenever the virus met a large proportion of pregnant women who were not immune In countries with rubella vaccination programs during childhood, where almost all women of child-bearing age are immune against the virus, CRS has become very rare—which could be

an interesting analogy to the apparent lack of clusters of microcephaly due to Zika virus in Africa and Asia

Although the initial suspicion of a

causal relationship was based on observed clusters of microcephaly trailing Zika virus outbreaks on the popula-tion level, we can already see other evidence supporting a causal relationship in regard to some of Austin Bradford Hill’s aspects This

is particularly true for temporality, biological plausibility, and analogy But clearly many gaps remain to be filled until we can decide

“that the most likely explanation is

EMBO Molecular Medicine Association of Zika virus and microcephaly Christina Frank et al

306

causation” (Hill, 1965) Especially data from

prospective or retrospective cohort studies

in the affected countries are eagerly awaited

to discern the strength of association (or

lack thereof), and to address knowledge

gaps for some of the other aspects On the

ecological level, it will be critical whether

we will see clusters of microcephaly in the

countries that were only affected in late

2015/early 2016, and whether the so

far-unexplained absence of microcephaly

clus-ters in Africa and Asia can be elucidated

This Opinion only addresses the putative

link between Zika virus infection during

pregnancy and congenital microcephaly In

addition, there are hints of associated ocular

problems in newborns (Ventura et al, 2016),

somewhat analogous to CRS In French

Poly-nesia, reports of microcephaly describe more

variable congenital CNS malformations

Should causality between Zika virus and

microcephaly be established, this phenotypic

and comparatively observable characteristic

may just be a proxy for a whole syndrome

of congenital Zika virus infection, for which

the same aspects of evidence ought to be

checked The question whether Zika virus

can also cause Guillain–Barre´ syndrome as a

post-infectious sequel in non-congenital

infections should be considered separately

Because we cannot yet characterize the

association between Zika virus and

micro-cephaly as causal, that in itself is no reason

to shift the investigative focus to other

hypotheses Any other putative factors put

forward as demonstrating similarly lagged correlations with recognized clusters of microcephaly, such as exposures to certain insecticides or medications, must be put through the same rigorous process of judg-ing the evidence They can be considered in case–control and cohort studies as additional exposures, to either confirm or refute any role in the causation of microcephaly in the groups of women who are studied

We hope that applying the Bradford Hill aspects of evidence for epidemiological causation provides a useful checklist for current and future evidence for or against a causal association between Zika virus infection in pregnancy and congenital micro-cephaly Giving the ongoing epidemiological and molecular research, especially in the Americas, some of the questions that we raised in this Opinion may already be answered by the time this article is published

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